EP1858965B1 - Polymerverbindung für biomedizinische verwendung und biochipsubstrat mit einer derartigen polymerverbindung - Google Patents

Polymerverbindung für biomedizinische verwendung und biochipsubstrat mit einer derartigen polymerverbindung Download PDF

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EP1858965B1
EP1858965B1 EP06737993.3A EP06737993A EP1858965B1 EP 1858965 B1 EP1858965 B1 EP 1858965B1 EP 06737993 A EP06737993 A EP 06737993A EP 1858965 B1 EP1858965 B1 EP 1858965B1
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Prior art keywords
polymer compound
group
ethylenically unsaturated
polymerizable monomer
unsaturated polymerizable
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French (fr)
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EP1858965A2 (de
EP1858965A4 (de
Inventor
Mitsutaka SUMITOMO BAKELIKE COMP. LTD. MATSUMOTO
Sumio Sumitomo Bakelite Company Ltd. SHIBAHARA
Takayuki SUMITOMO BAKELITE COMPANY LTD MATSUMOTO
Kanehisa SUMITOMO BAKELITE COMPANY LTD. YOKOYAMA
Sohei SUMITOMO BAKELITE COMPANY LTD. FUNAOKA
Daisuke SUMITOMO BAKELITE COMPANY LTD. MASUDA
Michael Patrick Coleman
Dominic Zichi
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Sumitomo Bakelite Co Ltd
Somalogic Inc
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Sumitomo Bakelite Co Ltd
Somalogic Inc
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/16—Nitrogen-containing compounds
    • C08K5/20—Carboxylic acid amides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
    • C08F220/28—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety
    • C08F220/285—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety and containing a polyether chain in the alcohol moiety
    • C08F220/286—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety and containing a polyether chain in the alcohol moiety and containing polyethylene oxide in the alcohol moiety, e.g. methoxy polyethylene glycol (meth)acrylate
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/04—Coating
    • C08J7/0427—Coating with only one layer of a composition containing a polymer binder
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/04—Coating
    • C08J7/043—Improving the adhesiveness of the coatings per se, e.g. forming primers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L23/00—Compositions of homopolymers or copolymers of unsaturated aliphatic hydrocarbons having only one carbon-to-carbon double bond; Compositions of derivatives of such polymers
    • C08L23/02—Compositions of homopolymers or copolymers of unsaturated aliphatic hydrocarbons having only one carbon-to-carbon double bond; Compositions of derivatives of such polymers not modified by chemical after-treatment
    • C08L23/04—Homopolymers or copolymers of ethene
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
    • C08F220/30—Esters containing oxygen in addition to the carboxy oxygen containing aromatic rings in the alcohol moiety
    • C08F220/305—Esters containing oxygen in addition to the carboxy oxygen containing aromatic rings in the alcohol moiety and containing a polyether chain in the alcohol moiety
    • C08F220/306—Esters containing oxygen in addition to the carboxy oxygen containing aromatic rings in the alcohol moiety and containing a polyether chain in the alcohol moiety and polyethylene oxide chain in the alcohol moiety
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
    • C08F220/36—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate containing oxygen in addition to the carboxy oxygen, e.g. 2-N-morpholinoethyl (meth)acrylate or 2-isocyanatoethyl (meth)acrylate
    • C08F220/365—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate containing oxygen in addition to the carboxy oxygen, e.g. 2-N-morpholinoethyl (meth)acrylate or 2-isocyanatoethyl (meth)acrylate containing further carboxylic moieties
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2365/00—Characterised by the use of macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain; Derivatives of such polymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2443/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing boron, silicon, phosphorus, selenium, tellurium or a metal; Derivatives of such polymers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/31504—Composite [nonstructural laminate]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/31504—Composite [nonstructural laminate]
    • Y10T428/31855—Of addition polymer from unsaturated monomers
    • Y10T428/31938—Polymer of monoethylenically unsaturated hydrocarbon

Definitions

  • the present invention relates to a polymer compound having a function of fixing a biologically active substance.
  • the present invention further relates to a surface coating material containing such a polymer compound, and relates to a biochip substrate using such a polymer compound.
  • proteomics can provide further information about the biological function of cells.
  • Proteomics includes qualitative and quantitative measurement of the gene activity by detecting and quantifying the expression on a protein level rather than a gene level.
  • Proteomics also includes a study of events which are not coded for gene, such as a post-translational modification of protein and an interaction between proteins.
  • Protein chip is a collective term used to refer to any device in which protein or a molecule for catching such a protein is fixed on a surface of a chip (a fine substrate or particle).
  • the protein chips at present are generally developed as an extension of DNA chips. Therefore, attempts are made in order to fix a protein or a molecule for catching such a protein on a surface of a chip such as glass substrate or particle (e.g. Japanese Patent Application Laid-open No. 2001-116750 ).
  • adsorption preventing agents are coated in order to prevent a nonspecific adsorption of the secondary antibody after the protein is fixed.
  • the ability of these agents for preventing the nonspecific adsorption is not sufficient.
  • the adsorption preventing agent is coated after the primary antibody is fixed, the coating is made on the fixed protein which prevents the reaction between the biochip and the secondary antibody.
  • EP 0 468 585 A2 relates to biologically active reagents prepared by using polymeric particles, analytical elements containing such reagents, and immunoassays and specific binding analytical methods using them.
  • WO 2004/039854 relates to polymeric support systems and, in particular, a macroporous plastic bead material containing a copolymer of crosslinked (meth)acrylate plastic. Park et al. (Anal. Chem. 76 (2004) : 2649-2655 ) describe the evaluation of 2-methacryloyloxyethyl phosphorylcholine polymeric nanoparticles for immunoassay of C-reactive protein detection.
  • WO 03/040700 relates to a water-swellable hydrophobic hydrogel, analytical devices incorporating the hydrogel, methods of using the hydrogel to prepare the analytical devices, and methods of using the analytical devices to detect, quantitate and discriminate between analytes in a sample.
  • An object of the present invention is to provide a polymer compound for biomedical use, which has an excellent ability of fixing biologically active substances, and hardly adsorbs to proteins nonspecifically, and has a chemical and physical stability against dissolving or deteriorating in the washing process, and can be suitably coated also on a surface of plastic substrates, as well as to provide a biochip substrate with a high S/N ratio using such a polymer compound.
  • a polymer compound obtained by copolymerizing an ethylenically unsaturated polymerizable monomer (a) having an alkylene glycol residue, an ethylenically unsaturated polymerizable monomer (b) having a functional group as specified in claim 1 for fixing a biologically active substance and an ethylenically unsaturated polymerizable monomer (c) as specified in claim 1 having a cross-linkable functional group has an excellent ability of fixing biologically active substances, and hardly causes a nonspecific adsorption, and can be coated uniformly on a plastic substrate without warping or waving, so that such a polymer compound can be used suitably for biochips. From this finding, the inventors have been accomplished the present invention.
  • a polymer compound for biomedical use which has an excellent ability of fixing biologically active substances, and hardly adsorbs to proteins nonspecifically, and has a chemical and physical stability against dissolving or deteriorating in the washing process, and can be suitably coated also on a surface of plastic substrates. It is also possible to provide a biochip substrate with a high S/N ratio using such a polymer compound.
  • the polymer compound of the present invention is characterized in that it can be obtained by copolymerizing an ethylenically unsaturated polymerizable monomer (a) having an alkylene glycol residue, an ethylenically unsaturated polymerizable monomer (b) having a functional group as specified in claim 1 for fixing a biologically active substance, and an ethylenically unsaturated polymerizable monomer (c) as specified in claim 1 having a cross-linkable functional group.
  • This polymer compound is a polymer having a nature of inhibiting a nonspecific adsorption of biologically active substances, a nature of fixing biologically active substances and a nature of cross-linking polymer chains.
  • the alkylene glycol residue acts to inhibit the nonspecific adsorption of biologically active substances
  • the functional group for fixing a biologically active substance acts to fix biologically active substances.
  • the ethylenically unsaturated polymerizable monomer (a) having an alkylene glycol residue to be used in the present invention is not limited to any special structure, but preferably may be a compound represented by the general formula [1] including a chain of (meth)acrylic group and alkylene glycol residue X having 1 to 10 carbon atoms.
  • alkylene glycol residue means an "alkyleneoxy group” (-R-O- wherein R is alkylene group) which remains after a condensation reaction of one or both end hydroxy groups of an alkylene glycol (HO-R-OH, wherein R is alkylene group) with other compounds.
  • alkylene glycol residue of methylene glycol (HO-CH 2 -OH) is methyleneoxy group (-CH 2 -O-)
  • alkylene glycol residue of ethylene glycol (HO-CH 2 -CH 2 -OH) is ethyleneoxy group (-CH 2 -CH 2 -O-).
  • the carbon number of the alkylene glycol residue X is 1 to 10, preferably 1 to 6, more preferably 2 to 4, still more preferably 2 to 3, the most preferably 2.
  • the repeating number p of the alkylene glycol residue is an integer from 1 to 100, preferably an integer from 2 to 100, more preferably an integer from 2 to 95, most preferably an integer from 20 to 90. In the case that the repeating number is not less than 2 but not more than 100, the carbon numbers of the alkylene glycol residue X repeated in a chain may be the same or may be different.
  • Examples of the ethylenically unsaturated polymerizable monomer (a) having the alkylene glycol residue include: methoxypolyethyleneglycol(meth)acrylate; (meth)acrylates of an ester substituted by one hydroxy group such as 2-hydroxyethyl(meth)acrylate, 2-hydroxypropyl(meth)acrylate, and 2-hydroxybutyl(meth)acrylate; glycerolmono(meth)acrylate; (meth)acrylate having a polypropyleneglycol side chain; 2-methoxyethyl(meth)acrylate; 2-ethoxyethyl(meth)acrylate; methoxydiethyleneglycol(meth)acrylate; ethoxydiethyleneglycol(meth)acrylate; ethoxypolyethyleneglycol(meth)acrylate and so on. Among them, methoxypolyethyleneglycol methacrylate is preferable in view of availability. In the present invention, "(me
  • the ethylenically unsaturated polymerizable monomer (b) having a functional group for fixing a biologically active substance according to the present invention is a compound represented by the following general formula [2] in which a (meth)acrylic group and an active ester group are bonded via an alkyl group or a chain of an alkylene glycol residue having 1 to 10 carbon atoms.
  • the carbon number of the alkylene glycol residue Y is 1 to 10, preferably 1 to 6, more preferably 2 to 4, still more preferably 2 to 3, the most preferably 2.
  • the repeating number q of alkylene glycol residue Y is an integer from 1 to 20, preferably an integer from 2 to 18, more preferably an integer from 3 to 16, the most preferably an integer from 4 to 14. If the repeating number is not less than 2 and not more than 20, the carbon numbers of the alkylene glycol residues repeated in a chain may be the same or may be different.
  • the “active ester group” means an ester group activated relative to a nucleophilic reaction by having a high acidic electron attracting group as one substituent of the ester group, selected from the group including p-nitrophenyl active ester group, N-hydroxysuccinimide active ester group, succinimide active ester group, phthalic acid imide active ester group, 5-norbornene-2,3-dicarboxyimide active ester group. Among them, p-nitrophenyl active ester group or N-hydroxysuccinimide active ester group is preferable, and p-nitrophenyl active ester group is the most preferable.
  • the preferable ratio of the ethylenically unsaturated polymerizable monomer (b) having a functional group for fixing a biologically active substance according to the present invention is 1 to 50 mol %, more preferably 1 to 30 mol %, the most preferably 1 to 20 mol %.
  • the ethylenically unsaturated polymerizable monomer (c) having a cross-linkable functional group according to the present invention is not limited to any special kind, insofar as the reaction of the cross-linkable functional group does not proceed during the synthesis of the polymer compound.
  • the cross-linkable functional group is a functional group generating a silanol group via hydrolysis, in view of its cross-linking ability in a lower temperature environment.
  • the functional group generating a silanol group via hydrolysis means a group which is easily hydrolyzed and generates a silanol group, when it comes into contact with water.
  • the ethylenically unsaturated polymerizable monomer having the functional group generating a silanol group via hydrolysis is an ethylenically unsaturated polymerizable monomer represented by the general formula [3] in which a (meth)acrylic group and an alkoxy silyl group are bonded to each other directly or via an alkyl chain having 1 to 20 carbon atoms.
  • Examples of the ethylenically unsaturated polymerizable monomer include 3-(meth)acryloxy propyl dimethyl methoxy silane, 3-(meth)acryloxy propyl dimethyl ethoxy silane, 3-(meth)acryloxy propyl methyl dimethoxy silane, 3-(meth)acryloxy propyl methyl diethoxy silane, 3-(meth)acryloxy propyl trimethoxy silane, 3-(meth)-acryloxy propyl triethoxy silane, 3-(meth)acryloxy propyl tris(methoxyethoxy) silane, 8-(meth)acryloxy octanyl trimethoxy silane, 11-(meth)acryloxy undecyl trimethoxy silane and so on.
  • 3-methacryloxy propyl trimethoxy silane, 3-methacryloxy propyl triethoxy silane, 3-methacryloxy propyl dimethyl methoxy silane and 3-methacryloxy propyl dimethyl ethoxy silane are preferable, in view of superior copolymerization ability relative to the ethylenically unsaturated polymerizable monomer having an alkylene glycol residue, or in view of the availability.
  • These ethylenically unsaturated polymerizable monomers each having an alkoxy silyl group may be used solely or as a combination of two or more kinds.
  • the preferable ratio of the ethylenically unsaturated polymerizable monomer (c) having a cross-linkable functional group according to the present invention is 1 to 20 mol %, more preferably 2 to 15 mol %, the most preferably 2 to 10 mol %.
  • the polymer compound according to the present invention may include another ethylenically unsaturated polymerizable monomer having a group other than the aforementioned group, in addition to the ethylenically unsaturated polymerizable monomer having an alkylene glycol residue, the ethylenically unsaturated polymerizable monomer having a functional group for fixing a biologically active substance and the ethylenically unsaturated polymerizable monomer having a cross-linkable functional group.
  • an ethylenically unsaturated polymerizable monomer (d) having an alkyl group may be copolymerized.
  • an ethylenically unsaturated polymerizable monomer (d) having an alkyl group n-butyl methacrylate, n-dodecyl methacrylate or n-octyl methacrylate is preferable.
  • a method of synthesizing the polymer compound of the present invention is not limited to any special method. From the viewpoint of easiness in the synthesis, however, it may be preferably a method of conducting a radical polymerization of a mixture containing, at least, the ethylenically unsaturated polymerizable monomer (a) having an alkylene glycol residue, the ethylenically unsaturated polymerizable monomer (b) having a functional group for fixing a biologically active substance, and the ethylenically unsaturated polymerizable monomer (c) having a cross-linkable group, under the existence of a polymerization initiator, in solvent.
  • the solvent is not limited to any special solvent insofar as each ethylenically unsaturated polymerizable monomer can be dissolved therein, but may be methanol, ethanol, t-butyl alcohol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform. These solvents may be used solely, or may be used as a combination of two or more kinds. In the case that the polymer compound is applied onto a plastic substrate, ethanol or methanol is preferable, in view of avoiding the denaturation of the substrate.
  • the polymerization initiator may be any usual radical initiator, including azo compounds such as 2,2'-azobisisobutyronitrile (hereinafter abbreviated as AIBN) and 1,1'-azobis(cyclohexane-1-carbonitrile); and organic peroxides such as benzoyl peroxide and lauryl peroxide; and so on.
  • AIBN 2,2'-azobisisobutyronitrile
  • 1,1'-azobis(cyclohexane-1-carbonitrile) 1,1'-azobis(cyclohexane-1-carbonitrile
  • organic peroxides such as benzoyl peroxide and lauryl peroxide
  • the chemical structure of the polymer compound of the present invention is not limited to any special structure insofar as the polymer compound is a copolymer obtained by copolymerizing at least the ethylenically unsaturated polymerizable monomers each having an alkylene glycol residue, a functional group for fixing a biologically active substance and a cross-linkable functional group, regardless of copolymer type such as random copolymer, block copolymer and graft copolymer type.
  • the molecular weight of the polymer compound of the present invention is preferably not less than 5000, more preferably not less than 10000, on the basis of number-average molecular weight, in view of easiness in the purification by separating the polymer compound from the unreacted ethylenically unsaturated polymerizable monomers.
  • the polymer compound of the present invention Owing to the polymer compound of the present invention, it is easy to provide a nature of restricting a nonspecific adsorption of biologically active substances, and a nature of fixing a specific biologically active substance, by coating a surface of a supporting substrate with the polymer compound. Furthermore, since the polymer compound of the present invention has a nature of cross-linking a plurality of polymer main chains, the cross-linking can be caused after the surface of the supporting substrate is coated. Thereby, the polymer compound on the substrate can have insolubility, so that signal degradation due to the supporting substrate washing can be reduced.
  • the coating of the polymer compound onto the surface of the supporting substrate may be achieved by the steps including (i) preparing a polymer compound solution which is obtained by dissolving a polymer compound in an organic solvent so that the concentration becomes 0.05 to 10 weight percent, (ii) applying the polymer compound solution onto the surface of the supporting substrate by a known process such as dipping or blowing, and (iii) then drying the applied solution in a room temperature environment or in a heated temperature environment. After that, a plurality of polymer main chains is cross-linked by any method suitable for a cross-linkable functional group.
  • the cross-linkable functional group is a functional group which generates a silanol group via hydrolysis
  • a mix solution obtained by containing water in an organic solvent may be used.
  • the contained water causes a hydrolysis, by which a silanol group is generated in the synthesized polymer.
  • a plurality of main chains is bonded, so that the polymer compound becomes insoluble.
  • the water content can be about 0.01 to 15 weight percent.
  • the organic solvent may be a simple solvent such as ethanol, methanol, t-butyl alcohol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, acetone, methylethylketone, or may be a solvent mixture thereof.
  • ethanol and methanol are preferable, because they do not cause denaturation of the plastic substrate, and they are easy to dry.
  • ethanol and methanol are preferable, because they can be mixed with water at a desirable ratio when the polymer compound is to be hydrolyzed in the solution.
  • the silanol groups in the polymer compound generate cross-linkings by condensing with silanol groups, hydroxy groups, amino groups in another polymer compound so as to dehydrate water. Furthermore, also in the case that hydroxy groups, carbonyl groups, amino groups exist on the surface of the supporting substrate, the condensation with dehydration is caused, so that a chemical bond with the surface of the supporting substrate can be made. Since the covalent bond which is formed by the condensation of the silanol group with dehydration is hardly hydrolyzed in its nature, the polymer compound coated on the surface of the supporting substrate hardly dissolves, or hardly detaches from the supporting substrate.
  • a temperature range in which the polymer compound is not thermally denaturated is preferable.
  • a heat treatment is preferably performed in a temperature range from 60 to 120 °C, for a time range from 5 minutes to 24 hours.
  • a material for the biochip substrate to be used in the present invention may be glass, plastic, metal and other materials, but plastic is preferable, in view of easiness in the surface treatment and the mass productivity. Thermoplastic is more preferable.
  • thermoplastic a less fluorescent thermoplastic is preferable.
  • a linear polyolefin such as polyethylene or polypropylene; a cyclic polyolefin; fluorine-contained resin and so on are preferably used.
  • saturated cyclic polyolefin is more preferably used, because of its excellency in heat resistance, chemical resistance, low fluorescence and moldability.
  • the saturated cyclic polyolefin means a saturated polymer obtained by hydrogenating a simple polymer having a cyclic olefin structure or by hydrogenating a copolymer of a cyclic olefin and an a -olefin.
  • An activating method may be a method of conducting a plasma treatment under a condition such as oxygen atmosphere, argon atmosphere, nitrogen atmosphere, or air atmosphere, or may be a method of conducting a treatment with excimer laser such as ArF or KrF. Particularly, the method of conducting a plasma treatment in an oxygen atmosphere is preferable.
  • the polymer compound of the present invention By applying the polymer compound of the present invention onto the supporting substrate, it is possible to produce a biochip substrate capable of restricting nonspecific adsorption of biologically active substances. Furthermore, by cross-linking the polymer compound, it is possible to provide the insolubility to the polymer compound on the supporting substrate. Thus, the supporting substrate coated by the polymer compound can be suitably used for a biochip use.
  • the biologically active substance to be fixed may be nucleic acid, aptamer, protein, oligopeptide, sugar chain, glycoprotein and so on.
  • nucleic acid it is preferable to introduce amino group in order to improve the reactivity with active ester group.
  • An introduction position where the amino group is to be introduced may be an end of a molecular chain or may be a side chain (also called "branch"). However, it is preferable that the amino group is introduced at an end of a molecular chain.
  • a method of attaching a droplet of the solution or dispersion of biologically active substances is preferable.
  • the biochip substrate After attaching the droplet, the biochip substrate is left at rest, so that the biologically active substance is fixed.
  • the biologically active substance For example, in the case that aminated nucleic acid is used, it is possible to fix the aminated nucleic acid by leaving it at rest for 1 hour at a temperature in a range from a room temperature to 80°C. The higher process temperature is preferable.
  • the liquid in which the biologically active substance is dissolved or dispersed is preferably alkaline.
  • the inactivation is preferably conducted with an alkali compound or a compound having a primary amino group in the case of active ester or aldehyde group.
  • alkali compound which can be preferably used include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium hydrogencarbonate, sodium dihydrogen phosphate, calcium hydroxide, magnesium hydroxide, sodium borate, lithium hydroxide, potassium phosphate.
  • Examples of the compound having a primary amino group which can be preferably used include methyl amine, ethyl amine, butyl amine, glycine, 9-aminoaquagene, aminobutanol, 4-aminobutyric acid, aminocaprylic acid, aminoethanol, 5-amino-2,3-dihydro-1,4-pentanol, aminoethanethiol hydrochloride, aminoethanethiol sulfuric acid, 2-(2-aminoethylamino)ethanol, 2-aminoethyl dihydrogen phosphate, aminoethyl hydrogensulfate, 4-(2-aminoethyl) morpholine, 5-aminofluorescein, 6-aminohexanoic acid, aminohexyl cellulose, p-aminohippuric acid, 2-amino-2-hydroxymethyl-1,3-propanediol, 5-aminoisophthalic acid, aminomethane, aminophenol, 2-
  • the biochip thus obtained by fixing the biologically active substance can be used for various analysis systems including an immunodiagnosis system, a gene microarray system, a protein microarray system, and a microfluidic device.
  • Polyethylene glycol methylether methacrylate also known as methoxypolyethylene glycol methacrylate
  • PEGMA methoxypolyethylene glycol methacrylate
  • MEONP p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate
  • MPDES 3-methacryloxypropyldimethylethoxy silane
  • Polyethylene glycol methylether methacrylate also known as methoxypolyethylene glycol methacrylate
  • PEGMA methoxypolyethylene glycol methacrylate
  • MEONP p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate
  • MPDES 3-methacryloxypropyldimethylethoxy silane
  • PEGMA methoxypolyethylene glycol methacrylate
  • MEONP p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate
  • MPDES 3-methacryloxypropyldimethylethoxy silane
  • PEGMA methoxypolyethylene glycol methacrylate
  • MEONP p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate
  • MPDES 3-methacryloxypropyldimethylethoxy silane
  • a saturated cyclic polyolefin resin (MFR (Melt Flow Rate) was 21 g/10 min, hydrogenation ratio was substantially 100 %, thermal deformation temperature was 123 °C), obtained via ring-opening polymerization of 5-methyl-2-norbornene and hydrogenation the polymerized product, was formed into a slide glass shape (dimension: 76mm ⁇ 26mm ⁇ 1 mm) via injection molding, so that a solid phase substrate was made as a supporting substrate. The surface of the substrate was oxidized by a plasma process in an oxygen atmosphere.
  • MFR Melt Flow Rate
  • This solid substrate was dipped into a 0.3 weight percent ethanol solution of each polymer compound obtained by the synthesis examples 1 to 4, and heated and dried for 4 hours at 65°C, so that a layer containing a polymer compound comprising an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue, an ethylenically unsaturated polymerizable monomer having an active ester group and an ethylenically unsaturated polymerizable monomer having a cross-linkable functional group was introduced on the surface of the solid phase substrate.
  • a saturated cyclic polyolefin resin (MFR (Melt Flow Rate) was 21 g/10 min, hydrogenation ratio was substantially 100 %, thermal deformation temperature was 123 °C), obtained via ring-opening polymerization of 6-methyl-2-norbornene and hydrogenation the polymerized product, was formed into a slide glass shape (dimension: 76mm ⁇ 26mm ⁇ 1 mm) via injection molding, so that a solid phase substrate was made. The surface of the substrate was oxidized by a plasma process in an oxygen atmosphere.
  • MFR Melt Flow Rate
  • a saturated cyclic polyolefin resin (MFR (Melt Flow Rate) was 21 g/10 min, hydrogenation ratio was substantially 100 %, thermal deformation temperature was 123 °C), obtained via ring-opening polymerization of 5-methyl-2-norbornene and hydrogenation the polymerized product, was formed into a slide glass shape (dimension: 76mm ⁇ 26mm ⁇ 1 mm) via injection molding, so that a solid phase substrate was made. The surface of the substrate was oxidized by a plasma process in an oxygen atmosphere. This solid substrate was dipped into a 2 volume percent ethanol solution of 3-aminopropylmethoxy silane.
  • the substrate was washed by pure water, and heated for 2 hours at 45°C so as to introduce amino group. Furthermore, the substrate was dipped into 1 volume percent aqueous solution of glutaraldehyde, and then washed by pure water, so that aldehyde group was introduced.
  • An amine-reactive slide glass substrate was made according to Example X in Japanese Patent Application Kohyo (Laid-Open under national phase of PCT Application) No. 2004-531390 .
  • 0.1 mol/L ethanol amine available from Wako Pure Chemical Industries, Ltd., ultrapure grade
  • 0.1 mol/L tris buffers available from SIGMA
  • one substrate according to Comparative Example 1 was subjected to the adsorption preventing treatment by dipping it into a quadruple dilution of a commercially available anti-adsorption agent, "block ace” (available from Dainippon Pharma Co., Ltd.), for 2 hours, in which PBS buffer (available from Nissui Pharma Co., Ltd.: a buffer obtained by dissolving 9.6g of Dulbecco PBS (-) for culturing tissue into 1L of pure water) was used as the diluent.
  • PBS buffer available from Nissui Pharma Co., Ltd.: a buffer obtained by dissolving 9.6g of Dulbecco PBS (-) for culturing tissue into 1L of pure water
  • Another substrate according to Comparative Example 1 was prepared without the adsorption preventing treatment.
  • the substrate according to Comparative Example 2 was subjected to the adsorption preventing treatment by dipping it into a quadruple dilution of the commercially available anti-adsorption agent, "block ace” (available from Dainippon Pharma Co., Ltd.), for 2 hours, in which PBS buffer (available from Nissui Pharma Co., Ltd.: a buffer obtained by dissolving 9.6g of Dulbecco PBS (-) for culturing tissue into 1L of pure water) was used as the diluent.
  • PBS buffer available from Nissui Pharma Co., Ltd.: a buffer obtained by dissolving 9.6g of Dulbecco PBS (-) for culturing tissue into 1L of pure water
  • a FBS (Fetal Bovine Serum) solution was prepared by diluting to 10 % with PBS buffer (available from Nissui Pharma Co., Ltd.: a buffer obtained by dissolving 9.6g of Dulbecco PBS (-) for culturing tissue into 1L of pure water).
  • PBS buffer available from Nissui Pharma Co., Ltd.: a buffer obtained by dissolving 9.6g of Dulbecco PBS (-) for culturing tissue into 1L of pure water.
  • the mouse IgG2a as antigen was added, so that 20 nmol/L solution was prepared.
  • This solution was diluted with 10 % FBS solution, which was diluted with PBS buffer (available from Nissui Pharma Co., Ltd.: a buffer obtained by dissolving 9.6g of Dulbecco PBS (-) for culturing tissue into 1 L solvent), so that 1 time diluted, 2 times diluted, 3 times diluted and 4 times diluted solutions were prepared.
  • PBS buffer available from Nissui Pharma Co., Ltd.: a buffer obtained by dissolving 9.6g of Dulbecco PBS (-) for culturing tissue into 1 L solvent
  • Antigen-antibody reactions were conducted by bringing these diluted solutions, as well as 10 % FBS solution containing no IgG2a, into contact with each substrate for 2 hours at 37°C.
  • the substrates were washed, for 5 minutes at a room temperature, with 1 ⁇ SSC buffer (obtained by diluting SSC20 ⁇ buffer available from Zymed Laboratories, Inc.) to which 0.05 wt % non-ionic surfactant, "Tween 20" (available from Roche Diagnostics K.K.) was added.
  • 1 ⁇ SSC buffer obtained by diluting SSC20 ⁇ buffer available from Zymed Laboratories, Inc.
  • Tween 20 available from Roche Diagnostics K.K.
  • biotin-labeled anti-mouse IgG2a as secondary antibody was added to PBS buffer (available from Nissui Pharma Co., Ltd.: a buffer obtained by dissolving 9.6g of Dulbecco PBS (-) for culturing tissue into 1L of pure water), so that 20 nmol/L solution was prepared.
  • PBS buffer available from Nissui Pharma Co., Ltd.: a buffer obtained by dissolving 9.6g of Dulbecco PBS (-) for culturing tissue into 1L of pure water
  • the substrates were washed, for 5 minutes at a room temperature, with 1 ⁇ SSC buffer (obtained by diluting SSC20 ⁇ buffer available from Zymed Laboratories, Inc.) to which 0.05 wt % non-ionic surfactant, "Tween 20" (available from Roche Diagnostics K.K.) was added.
  • 1 ⁇ SSC buffer obtained by diluting SSC20 ⁇ buffer available from Zymed Laboratories, Inc.
  • Tween 20 available from Roche Diagnostics K.K.
  • each substrate was washed , for 5 minutes at a room temperature, with 1 ⁇ SSC buffer (obtained by diluting SSC20 ⁇ buffer available from Zymed Laboratories, Inc.) to which 0.05 wt % non-ionic surfactant, "Tween 20" (available from Roche Diagnostics K.K.) was added, so that each substrate was labeled.
  • SSC buffer obtained by diluting SSC20 ⁇ buffer available from Zymed Laboratories, Inc.
  • Tween 20 available from Roche Diagnostics K.K.
  • each measurement of the fluorescent amount in Examples and Comparative Examples was conducted by means of a microarray scanner, "ScanArray” (available from Packard BioChip Technologies).
  • the laser power was 90 %
  • the PMT sensitivity was 50 %
  • the excitation wavelength was 649 nm
  • the measurement wavelength was 670 nm
  • the resolution was 50 ⁇ m.
  • biochip substrates according to the present invention have the lower background values and the higher signal intensity values, in comparison with a case that a conventional aldehyde substrate was treated with a commercially available anti-adsorption agent.
  • biochip substrates according to the present invention have the lower background values than that of the substrate according to Japanese Patent Application Kohyo (Laid-Open under national phase of PCT Application) No. 2004-531390 . It means that nonspecific protein contained in serum hardly adheres to the biochip substrates according to the present invention. Also, it was understood that the biochip substrates according to the present invention have the lower signal intensity value of the primary antibody spotted part without any antigen. It means that the primary antibody is fixed on the substrate in a condition that the antibody function thereof is active. Also, it was understood that the biochip substrates according to the present invention exhibit excellent reproducibility.
  • 0.1 mol/L ethanol amine available from Wako Pure Chemical Industries, Ltd., ultrapure grade
  • T5912 available from SIGMA
  • Fluorescent amount measurement was conducted for each Experiment, so that the spot signal intensity value and the background value were evaluated.
  • each measurement of the fluorescent amount was conducted by means of a microarray scanner, "ScanArray” (available from Packard BioChip Technologies).
  • the laser power was 90 %
  • the PMT sensitivity was 50 %
  • the excitation wavelength was 649 nm
  • the measurement wavelength was 670 nm
  • the resolution was 50 ⁇ m.

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Claims (13)

  1. Polymerverbindung, die durch Copolymerisieren eines ethylenisch ungesättigten, polymerisierbaren Monomers (a), das einen Alkylenglykolrest aufweist, eines ethylenisch ungesättigten, polymerisierbaren Monomers (b), das eine funktionelle Gruppe zum Fixieren einer biologisch aktiven Substanz aufweist, und eines ethylenisch ungesättigten, polymerisierbaren Monomers (c), das eine vernetzbare, funktionelle Gruppe aufweist, und gegebenenfalls eines ethylenisch ungesättigten, polymerisierbaren Monomers (d), das eine Alkylgruppe aufweist, erhalten wird,
    wobei das ethylenisch ungesättigte, polymerisierbare Monomer (b), das eine funktionelle Gruppe zum Fixieren einer biologisch aktiven Substanz aufweist, ein Monomer mit einem aktiven Ester ist, repräsentiert durch die nachstehende allgemeine Formel [2],
    Figure imgb0013
     worin R3 ein Wasserstoffatom oder eine Methylgruppe ist; Y eine Alkylgruppe oder ein Alkylenglykolrest mit 1 bis 10 Kohlenstoffatomen ist; W eine aktive Estergruppe, ausgewählt aus der Gruppe, bestehend aus p-Ni-trophenylestergruppe, N-Hydroxysuccinimidestergruppe, Succinimidestergruppe, Phthalsäureimidestergruppe und 5-Norbornen-2,3-dicarboxyimid-estergruppe, ist; q eine ganze Zahl von 1 bis 20 ist; in einem Fall, bei dem q eine ganze Zahl von nicht weniger als 2 und nicht mehr als 20 ist, das wiederholte Y gleich oder verschieden sein kann, und
    wobei das ethylenisch ungesättigte, polymerisierbare Monomer (c), das eine vernetzbare, funktionelle Gruppe aufweist, ein Monomer mit einer Alkoxysilylgruppe ist, repräsentiert durch die nachstehende allgemeine Formel [3],
    Figure imgb0014
     worin R4 ein Wasserstoffatom oder eine Methylgruppe ist; Z eine Alkylgruppe mit 1 bis 20 Kohlenstoffatomen ist; mindestens eine der Gruppen A1, A2, A3 eine hydrolysierbare Alkoxygruppe ist und der Rest von A1, A2, A3 (eine) Alkylgruppe(n) sind.
  2. Polymerverbindung gemäß Anspruch 1, wobei das ethylenisch ungesättigte, polymerisierbare Monomer (a), das einen Alkylenglykolrest aufweist, ein Monomer ist, das durch die nachstehende allgemeine Formel [1] repräsentiert wird,
    Figure imgb0015
     worin R1 ein Wasserstoffatom oder eine Methylgruppe ist; R2 ein Wasserstoffatom oder eine Alkylgruppe mit 1 bis 20 Kohlenstoffatomen ist; X ein Alkylenglykolrest mit 1 bis 10 Kohlenstoffatomen ist; p eine ganze Zahl von 1 bis 100 ist; in einem Fall, bei dem p eine ganze Zahl von nicht weniger als 2 ist, das wiederholte X gleich oder verschieden sein kann.
  3. Polymerverbindung gemäß Anspruch 1, wobei das ethylenisch ungesättigte, polymerisierbare Monomer (a), das einen Alkylenglykolrest aufweist, Methoxypolyethylenglykolacrylat oder Methoxypolyethylenglykolmeth-acrylat ist.
  4. Polymerverbindung gemäß Anspruch 3, wobei eine durchschnittliche Wie-derholungsanzahl des Ethylenglykolrests des Methoxypolyethylenglykol-acrylats und/oder Methoxypolyethylenglykolmethacrylats 3 bis 100 beträgt.
  5. Polymerverbindung gemäß einem jeglichen der Ansprüche 1 bis 4, wobei der aktive Ester p-Nitrophenylester oder N-Hydroxysuccinimidester ist.
  6. Polymerverbindung gemäß einem jeglichen der Ansprüche 1 bis 5, wobei das ethylenisch ungesättigte, polymerisierbare Monomer (d), das eine Alkylgruppe aufweist, mindestens ein Monomer, ausgewählt aus n-Butyl-methacrylat, n-Dodecylmethacrylat, n-Octylmethacrylat und Cyclohexylmethacrylat, ist.
  7. Oberflächenbeschichtungsmaterial, das die Polymerverbindung gemäß einem jeglichen der Ansprüche 1 bis 6 umfasst.
  8. Biochipsubstrat, das durch Ausbilden einer Schicht, die die Polymerverbindung gemäß einem jeglichen der Ansprüche 1 bis 6 umfasst, auf einer Oberfläche eines Trägersubstrats erhalten wird.
  9. Biochipsubstrat gemäß Anspruch 8, wobei das Trägersubstrat aus Plastik hergestellt ist.
  10. Biochipsubstrat gemäß Anspruch 9, wobei das Plastik ein gesättigtes, cyclisches Polyolefin ist.
  11. Verfahren zum Herstellen des Biochipsubstrats gemäß einem jeglichen der Ansprüche 8 bis 10, umfassend die Arbeitsvorgänge:
    Auftragen einer Lösung, die die Polymerverbindung gemäß einem jeglichen der Ansprüche 1 bis 6 enthält, auf eine Oberfläche eines Trägersubstrats und
    nach dem Auftragungsarbeitsvorgang Vernetzen der Polymerverbindung.
  12. Biochip, der durch Fixieren einer biologisch aktiven Substanz an dem Biochipsubstrat gemäß einem jeglichen der Ansprüche 8 bis 10 erhalten wird.
  13. Biochip gemäß Anspruch 12, wobei die biologisch aktive Substanz mindestens eine Substanz, ausgewählt aus Nukleinsäure, Aptamer, Protein, Oligopeptid, Zuckerkette und Glykoprotein, ist.
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EP1176423A1 (de) * 2000-07-27 2002-01-30 BioChip Technologies GmbH Auf einer Oberfläche befestigte multifunktionelle Polymere-Netzwerke für Sensorchips

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US8293190B2 (en) 2012-10-23
CA2601432C (en) 2014-07-08
US20080175758A1 (en) 2008-07-24
EP1858965A2 (de) 2007-11-28
WO2006101798A3 (en) 2007-02-01
AU2006227805B2 (en) 2010-06-24
JP5552474B2 (ja) 2014-07-16
JP2006258458A (ja) 2006-09-28
JP2008533489A (ja) 2008-08-21
AU2006227805A1 (en) 2006-09-28
KR101268553B1 (ko) 2013-05-28
JP2012078365A (ja) 2012-04-19
EP1858965A4 (de) 2010-11-10
US8088340B2 (en) 2012-01-03
US20120070474A1 (en) 2012-03-22
WO2006101798A2 (en) 2006-09-28
KR20070110860A (ko) 2007-11-20
CA2601432A1 (en) 2006-09-28
JP5046050B2 (ja) 2012-10-10

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